Identification and Genomic Characterization of Parvovirus B19V Genotype 3 Viruses from Cases of Meningoencephalitis in West Bengal, India

ABSTRACT Brain infections are a major public health problem in India and other parts of the world, causing both mortality and lifelong disability. Even after a thorough investigation, many cases remain without an etiological diagnosis. Primate erythroparvovirus 1 (B19V) has been identified as a pathogen associated with undiagnosed meningoencephalitis in other settings, including the United Kingdom, France, and Latvia. Here, we reported 13/403 (3.2%) B19V PCR positive cases of meningoencephalitis in West Bengal, India. The positive samples were mostly from children (10/13, 76.92%) and presented as a spectrum consisting of acute encephalitis (7/13), acute meningoencephalitis (3/13), and meningitis (3/13). Of the 13 cases, 8/13 (61.5%) had no known etiology and 5/13 (38.5%) had a previous etiological diagnosis. The cases did not cluster in time or by location, suggesting sporadic occurrence rather than outbreaks. We were able to retrieve the complete B19V genomes from cerebrospinal fluid (CSF) in 12/13 cases. The sequences clustered into genotype 3b with complete genomes from Brazil, Ghana, and France, and partial genomes from India and Kyrgyzstan. This is the first report of B19V in cases of neurological infections from India. It highlights the need to evaluate the causal relationship between B19V with meningoencephalitis in the country. These were also the first complete genomes of genotype 3b from CSF and will be critical in the evaluation of the relationship between genotypes and disease. IMPORTANCE Cases of meningoencephalitis with no known etiology remain a major challenge to clinical management of brain infections across the world. In this study, we detected and characterized the whole-genome of primate erythroparvovirus 1 (B19V) in cases of meningoencephalitis in India. Our work highlighted the association between B19V and brain infections which has been reported in other countries. Our work also emphasized the need to examine the role of B19V in meningoencephalitis, specifically whether it caused or contributed to the disease together with other pathogens in India. Our study provided the first 12 genomes of B19V from cerebrospinal fluid. These genomes will contribute to an understanding of how the virus is changing across different locations and over time.

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Editor, Microbiology Spectrum Reviewer comments: Reviewer #1 (Comments for the Author): B19V is known to be associated with brain infections in other endemic settings, this work emphasizes the need to examine whether it causes or contributes to the disease together with other pathogens. The authors have attempted to correlate B19V Parvovirus genotype prevalence with the neurological disease, especially among children. Through phylogenetic cluster analyses, the authors have depicted that the cases are sporadic in nature, and not an incidence of any outbreak in India. This study is the first report on association of genotype 3b of B19V Parvovirus with meningoencephalitis in West Bengal, India. In this context, the report is important and significant. Also, the study provides 12 complete genomes of B19V from cerebrospinal fluid, which will be useful information for other studies. Moreover, it encompasses samples from pediatric age group showings signs of acute encephalitis, acute meningoencephalitis and meningitis. Not many studies highlight the clinical manifestations caused by B19V in the endemic settings of India. The manuscript is written well and sounds interesting. However, with only 13 positives samples, it is difficult to establish clear causal correlation.
Minor Comments 1. More number of reference strains around the world (spatial and temporally distributed) would have added strength in the phylogenetic analyses to reveal a better picture of ancestry of the 12 representative strains included in the study.
2. References should be updated.

Reviewer #3 (Comments for the Author):
The article by Pattabiraman decribed the identication of parvovirus B19, in samples of nucleic acids, isolated from CSF of patients that presented meningoencepahitis in West Bengal, India. From the 403 samples, only 13 tested positive for parvovirus B19, and in a few cases there was co-infection with other pathogens. The authors recovered full parvovirus B19 genomes and sequence them. The sequence were not identical but group togheter when compare to full genomes or partial genomes from Asia. This article rises concern about the posibility that parvovirus B19 could be a virus associated to the development of encephalitis, and sugest that future cases include testing for it. Major points that could be improved are: -The authors anly showed thre filogenetic tres and the supplementary data inludes the accesión number of the different full genomes sequences. As a reader I wonder what are he differences betwen all the genomes isolated, since the authors mentioned they are not identical. A description of the differences found could be included. -In the discussion, although it is recognized that parvovirus B19 could only be a bystandars agent and it is inicated that its posible role on develpomet of meningitis or other brain inflamations must be explore, it would be nice see this point with more information if there is some in the literature. Does parvovirus B19 infect cell that are in teh brain? Neurons, glia, endotelial cells? -The references are presented in diferente formats, this must be corrected, Just to mention the two more evient: reference 31 and 39 Several minor point that should be improved.

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Response to the reviewers
We thank the reviewers for accepting to review our manuscript and for their considered comments. Changes have been made to the manuscript based on these comments and they are addressed in line below -Reviewer #1 (Comments for the Author): Minor Comments 1. More number of reference strains around the world (spatial and temporally distributed) would have added strength in the phylogenetic analyses to reveal a better picture of ancestry of the 12 representative strains included in the study.
All complete and near complete genomes of Primate Erythroparvovirus 1 were included for the phylogenetic analysis (n=247). The details of location and collection dates are now provided in Appendix 1 and the methods sections has been modified to reflect this as follows -All available complete and near complete ( 4070-5596 bp) genome sequences (n=247) of Primate Erythroparvovirus 1 (B19V) were retrieved from the NCBI database. These represent sequences from across the world (Netherlands 65, Brazil 29, Germany 19, Finland 15, USA 13, Ghana 7, France 6, Serbia 5, China 2, Belgium 2, Vietnam 2, Kenya 1, Bulgaria 1, United Kingdom 1, data NA 79), where collection dates and release dates are available, release dates were between 1993-2021 and collection dates between 2002-2017 (Appendix 1).

References should be updated.
References have been reformatted and updated.

Reviewer #3 (Comments for the Author):
Major points that could be improved are: 1. -The authors anly showed thre filogenetic tres and the supplementary data inludes the accesión number of the different full genomes sequences. As a reader I wonder what are he differences betwen all the genomes isolated, since the authors mentioned they are not identical. A description of the differences found could be included. 2. -In the discussion, although it is recognized that parvovirus B19 could only be a bystandars agent and it is inicated that its posible role on develpomet of meningitis or other brain inflamations must be explore, it would be nice see this point with more information if there is some in the literature. Does parvovirus B19 infect cell that are in teh brain? Neurons, glia, endotelial cells?

This has been included in
The following paragraph has been added to the Discussion section Current evidence supported by cell culture experiments suggests that B19V has a strong tropism for cells of the erythroid lineage(12). Viral DNA has been detected in a wide range of tissues however it is not clear whether the infection in these tissues is productive(12). B19V has been detected in oligodendroglia in different parts of the brain in post-mortem tissue(44). These studies suggest that B19V can persist in the brain . Early observations suggest a possible role in the demyelination process and support an immune mediated pathophysiology (12,43,44).
3. -The references are presented in diferente formats, this must be corrected, Just to mention the two more evient: reference 31 and 39 The reference formats have been corrected Several minor point that should be improved.

-Importance section lines 55-56 must be rephrased.
The importance section has been rewritten as follows: Cases of meningoencephalitis with no known aetiology remain a major challenge to clinical management of brain infections across the world. In this study, we detected and characterized the whole genome of Primate Erythroparvovirus 1 (B19V) in cases of meningoencephalitis in India.
Our work highlights the association between B19V and brain infections which has been reported from other countries. Our work also emphasizes the need to examine the role of B19V in meningoencephalitis, specifically, whether it causes or contributes to the disease together with other pathogens in India. Our study provides the first 12 complete genomes of B19V from cerebrospinal fluid. These genomes will contribute to an understanding of how the virus is changing across different locations and over time.

7.-line 234 increase is writen twice
This line has been modified as follows -Serological data -a seropositivity of 30-50% , as well as a reported increase in positivity with age suggests that the virus is circulating in the population (7,11,38). Your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication. You will be notified when your proofs are ready to be viewed.
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